Fatma Matpan Bekler

Purification and characterization of thermostable and detergent-stable α-amylase from Anoxybacillus sp. AH1.

A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purified and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9% were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30% glycerol, retaining 85% of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 µmol and 0.929 µmol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. α-Amylase activity was enhanced by β-mercaptoethanol (β-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fluoride (PMSF) had a strong inhibitory effect on the amylase activity.

Isolation and production of thermostable α-amylase from thermophilic Anoxybacillus sp. KP1 from Diyadin hot spring in Ağri, Turkey

A novel amylolytic enzyme producing thermophilic bacterial strain KP1 from the Diyadin hot spring water in Ağri, Turkey, was isolated in the present study. Phylogenetic analysis based on the partial 16S rRNA gene, biochemical and physiological tests revealed that the strain KP1 belongs to the genus Anoxybacillus. The pH and temperature optima for the α-amylase production by Anoxybacillus sp. KP1 were 8.0 and 50°C, respectively, where the maximum growth was obtained at the 28th hour of incubation and the highest α-amylase activity was obtained at the 40th hour of incubation (8979.6 U/mL). The optimum pH and temperature for the enzyme activity were 8.0 and 60°C, respectively. The maximum α-amylase production was secreted in the presence of 2% (w/v) soluble starch (10837.7 U/mL). Among the various organic and inorganic nitrogen sources tested, while keeping the beef extract concentration constant, casamino acid (14310.6 U/mL), urea (14126 U/mL), and tryptone (13217.2 U/mL) at a concentration of 2% gave the maximum α-amylase production. The enzyme activity was enhanced in the presence of 1.5 mM Mn2+ (123%), whereas it was strongly inhibited 1.5 mM by Hg2+. Inhibition by 89% was obtained also with sodium dodecyl sulphate (1%). The enzyme was found to be relatively stable at a range of pH and temperature.

Isolation and characterization of long-chain alkane–degrading Acinetobacter sp. BT1A from oil-contaminated soil in Diyarbakır, in the Southeast of Turkey

ABSTRACT A strain of long-chain alkane–degrading bacteria, BT1A, was isolated from oil-contaminated soil in Diyarbakır, in the southeast of Turkey. Morphological, biochemical, and physiological characterization and 16S rRNA gene sequence analysis showed that the strain BT1A was a member of Acinetobacter genus, and it was found to be closely related to Acinetobacter baumannii. The strain BT1A was able to utilize crude petroleum as carbon and energy sources in order to grow. Among the aliphatic hydrocarbons, growth was observed only in the medium containing long-chain alkanes (tridecane, pentadecane, and hexadecane) and squalene. Hexadecane was the most preferred hydrocarbon among the long-chain alkanes. Gas chromatography–mass spectrometry (GC-MS) analysis showed that BT1A degraded 83% of n-alkanes of 1% crude oil in 7 days. The present study indicates that the isolated strain can well be used for biodegradation of hydrocarbons in oil-contaminated sites.

Anoxybacillus sp. AH1, an α-amylase-producing thermophilic bacterium isolated from Dargeçit hot spring

Abstract The present study was conducted to isolate α-amylase-producing thermophilic bacteria from Darge¸cit hot springs in Turkey. The morphological, biochemical and physiological characterisation, as well as genetic analysis by 16S rRNA sequences indicated that the isolated strain AH1 was a member of Anoxybacillus genus. The strain was aerobe, Gram-positive and spore-forming rod, exhibiting optimum growth temperature and pH of 60ºC and 7.0-7.5, respectively. Optimization of growth medium and enzyme assay conditions for extracellular α-amylase production by the novel thermophilic Anoxybacillus sp. AH1 were carried out in many different media containing a variety of carbon and nitrogen sources. Among various carbon and nitrogen sources, peptone (2054.1 U/mL) at 1% and maltose (1862.9 U/mL) at 0.5% increased α-amylase activity, compared to controls. Moreover, a high enzyme production was observed with potato starch at 0.5% and 1% (2668.4 U/mL and 3627 U/mL, respectively), as well as with 1% soluble starch (2051.9 U/mL). The enzyme activity was found to be rather high in the presence of CaCl2 up to 100 mM.

Thermophilic and Halophilic Microorganisms Isolated from Extreme Environments of Turkey, with Potential Biotechnological Applications

Turkey has a great number of different ecological areas, owning over 200 hot water resources and various hypersaline environments with a broad microbial diversity and opportunities for newly isolated microorganisms from extreme environments for many industrial applications. A variety of thermophilic and halophilic microorganisms in different regions of Turkey have been isolated and identified. The thermophilic bacterial members studied were Anoxybacillus, Geobacillus, Bacillus, Brevibacillus, and Aeribacillus belonging to the Bacillaceae family and the other thermophiles such as Thermus and Thermomonas, whereas the isolated halophilic microorganisms were mainly found to be members of the archaeal family Halobacteriaceae or grouped into bacterial phylum Bacteroidetes. In summary, the present study reviews on (1) isolating and identifying thermophiles and halophiles single or as community from various extreme habitats in Turkey based on conventional (morphological, physiological and biochemical tests) and/or molecular methods, (2) screening these extremophiles for industrially important enzymes, (3) studying other novel products and their use in other areas of biotechnology, and finally (4) discussing about the development strategies and the future perspectives on poorly studied extremophilic microorganisms in the country to fulfill future biotechnological and industrial demands.